The brush border (bb) Na+/H+ exchanger (NHE) is part of the neutral NaCl absorptive process which accounts for intestinal basal Na absorption and becomes abnormal in diarrheal diseases. NHE3 has been shown to be the functional intestinal bb isoform based on its amiloride resistance and inhibition by protein kinase C. A new observation shown by us is the contribution of NHE2 to intestinal Na absorption based on separation of NHE2 and NHE3 activity on intestinal bb Na+/H+ exchange using an amiloride analogue, H0E694. Therefore, we propose studies to define the contribution of NHE2 and NHE3 to small intestinal (rabbit ileum) and colonic (rat descending colon) bb Na+/H+ exchange under basal conditions and using model conditions to mimic changes in intestinal NaCl absorption and bb Na+/H+ exchange which occur in digestion and in diarrheal diseases (stimulation (epidermal growth factor) or by inhibition (carbachol via protein kinase C)). To further understand the biochemical and molecular mechanisms that physiologically modulate NHE2 activity, we propose to use three cell models, rabbit ileal bb NHE2, HT29/NHE2 and PS12O/NHE2 (NHE2 stably expressed in intestinal epithelial HT29/19A cells and NHE deficient PS 120 cells), to probe structure/function relationship of NHE2: (1) to study the role of NHE2 phosphorylation in regulating NHE2 activity by two dimensional phosphopeptide mapping with separating the cell surface and intracellular forms of NHE2 using biotinylation; (2) to determine whether the increase in V max of NHE2 by growth factors/protein kinases is due to an increase in the turnover number of cell surface NHE2 and/or an increase in the amount of cell surface exchangers; (3) to study the mechanism of NHE2 regulation by accessory proteins: (a) calmodulin (CaM): phosphorylation, turnover number and membrane shuffling of NHE2 will be probed as mechanisms of inhibition of NHE2 by CaM as a consequence of in vivo binding of CaM to NHE2 demonstrated by co-immunoprecipitation. The CaM binding site of NHE2 will be identified by truncation and site directed mutant studies. (b) p32 and p34: In vivo association of these two phosphoproteins to NHE2 has been established by co-immunoprecipitation. We propose to determine the identity of these two potential regulatory proteins; (4) to map the organization of growth factor/protein kinase regulator subdomains on the putative C-terminus of NHE2 by truncation mutant studies. These subdomains are potential sites for phosphorylation and/or binding of associated proteins which mediate growth factor/protein kinase regulation of NHE2.